Benzo[a]pyrene is a potent mutagen/carcinogen that is metabolically activated inside cells, including to its (+)-anti-7,8-diol-9,10-epoxide [(+)-anti-B[a]PDE], which gives DNA adducts, principally at N2-Gua. We showed that (+)-anti-B[a]PDE induces base substitution, frameshifts, insertion and deletions (supF gene of plasmid pUB3 in E. coli). The question we are addressing is: how is (+)-anti-B[a]PDE able to induce such a diverse array of mutations? Our working hypothesis is that adduct mutational complexity is due to adduct conformational complexity, and that the major adduct [(+)-trans-anti-B[a]P-N2-Gua] is able to induce the majority of these mutations. Adduct conformation--and, thereby, mutation-- may be controlled by various factors, notably, DNA sequence context. (1) We showed that little of mutagenesis can be attributed to AP sites formation from (+)-anti-B[a]PDE adducts. We showed that trans-(+)-anti- B[a]P-N2-Gua can be unstable in ds (but not ss) DNA and gives its corresponding tetraols. Although this unexpected reaction is interesting, it is unlikely to be at the root of any important mutational observations. (2) Using adduct site-specific methods, we showed previously that (+)- trans-anti-B[a]P-N2-Gua induced G->T mutations in a 5'-TG-3' sequence context, which correlated with our studies showing that (+)-anti-B[a]PDE itself induced principally G->T mutations in 5'-TG-3' sequence contexts suggesting a mechanistic link. 3. In supF (+)-anti-B[a]PDE induced mostly G->A in one sequence context (5'-CGT-3'). In preliminary experiments we show that (+)-trans-anti- B[a]P-N2-Gua also induces G->A mutations in this sequence context. 4. In supF we identified a sequence (5'-CGG-3') where the mutagenic specificity seemed to be changeable, being G->T in some cases and G->T, A & C in others. If our hypothesis is correct, then this might be a conformationally sensitive site. We now show that trans-(+)-anti-B[a]P- N2-Gua indeed can change its pattern of mutations in this sequence context; for example, the mutational pattern following PEG treatment (predominantly G->T) is different than following ethanol precipitation (a mixture of G->T, A & C mutations). Herein we propose to gather further evidence that adduct conformational complexity is at the root of adduct mutational complexity. We also seek to establish sequence contexts where (+)-trans-anti-B[a]P-N2-Gua can induce each individual mutation (e.g., G->T only, G->A only, and G->C only), as a prelude to doing structural studies to determine what conformation is responsible for what mutation and why. The initiation of parallel studies with shuttle vectors in human cells is also proposed.